Current methods for identifying an antibody or a target molecule involve laborious processes of isolating antibodies from activated human B-cells. For example, isolation of fully human antibodies from B-cells of immunized or cancer patients is considered as an advantageous route to fully human antibodies. Several companies provide commercial services for isolating single B-cells from humans. These B-cells are either immortalized or the genetic information of the immunoglobulins of the single cells is recovered. Such methods may involve laborious and expensive high-throughput techniques, including techniques for isolating genetic information cell by cell, immortalizing thousands of cells and screening their respective output on the target tissue.
Since recently several companies offer whole genome sequencing services, or machines that can be used to accomplish respective tasks. This includes include Roche's 454 system, Illuminas's Solexa system, and Helico Biosciences Heliscope system. Helico's, for example, can sequence 2×109 bases in 24 hours with a single machine by also keeping quantitative distribution of target sequences.
U.S. Pat. No. 7,288,249 discloses a method for identifying an antigen which is differentially expressed on the surface of two or more distinct cell populations. Immunization triggers B cells to make a VH-VL combination that binds the immunogen to proliferate (clonal expansion) and to secrete the corresponding antibody. The process according to U.S. Pat. No. 7,288,249 however involves cloning of the VH and VL genes (the VH and the VL genes are cloned separately by polymerase chain reaction (PCR)), and the VH and the VL genes are recombined randomly in phage libraries (i.e. there is no selection of the most abundant VH and VL genes), which is then searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Nucleic acid encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA is obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5′ and 3′ ends of rearranged VH and VL genes. To find useful antibodies, the naive antibody phage library is screened against live cancer cells.
U.S. Pat. No. 6,897,028 describes a method for identification of molecular targets in which a protein binds to a ligand, screening the ligand against a peptide or protein library wherein the peptide or protein members of the library are selected from expression products of a cDNA library derived from a cell and fragments of those expression products. The process also involves determining the nucleic acid sequence which encode the members which have been separated from the library and translating these nucleic acid sequences into peptide sequences and identifying the protein.
U.S. Publication No. 20060141532 (application Ser. No. 11/286,917) discloses methods for identifying and designing immunogenic peptides by using a protocol for determining the amino acid sequence of certain VH or VL regions of an anti-idiotypic antibody as disclosed in Iwasaki, et al. Eur. J. Immunol., 24:2874-2881, 1994. The amino acid sequence of the antigen is determined by standard amino acid analysis techniques or by chemical sequencing; the amino acid sequence of VH and/or VL regions of the anti-idiotypic antibody are determined by sequencing the genomic DNA or cDNA encoding the respective region according to techniques known in the art.
WO2005/094159 describes the isolation of binding peptides from immortalized lymphocytes and the testing of these binding peptides, usually immunoglobulins of the type IgM, for selective binding to tumor tissue, but not healthy tissue. The binding peptide may inhibit proliferation of tumors.
WO03/052416 describes an approach for the isolation and sequencing of candidate VH and VL sequences from B cells. The immunoglobulins encoded by said candidate VH and VL sequences are potentially useful in the treatment of infections. The methodology described in WO03/052416, as well as all others methods disclosed in the prior art, require the manipulation of the nucleic acids prior to sequencing. In particular time consuming cloning steps are necessary which make it impossible to practice the method in a scale as disclosed in the present to invention.
As is the case for WO03/052416, all the methods discussed above involve laborious process of isolating nucleic acids encoding a target and/or genomic sequencing by ultra high-throughput techniques. However, a less laborious, cost effective and/or easier method for identifying a target molecule by analyzing sequence data of the VH and VL chains and by determining the most dominant VH and VL would be advantageous.